Colorectal cancer (CRC) is a leading cause of cancer death, yet primary prevention remains the best approach to reducing overall morbidity and mortality. Studies have shown that COX-2–derived PGE2 promotes CRC progression, and both nonselective COX inhibitors (NSAIDs) and selective COX-2 inhibitors (such as glucocorticoids) reduce the number and size of colonic adenomas. However, increased gastrointestinal side effects of NSAIDs and increased cardiovascular risks of selective COX-2 inhibitors limit their use in chemoprevention of CRC. We found that expression of 11β–hydroxysteroid dehydrogenase type II (11βHSD2), which converts active glucocorticoids to inactive keto-forms, increased in human colonic and Apc+/min mouse intestinal adenomas and correlated with increased COX-2 expression and activity. Furthermore, pharmacologic inhibition or gene silencing of 11βHSD2 inhibited COX-2–mediated PGE2 production in tumors and prevented adenoma formation, tumor growth, and metastasis in mice. Inhibition of 11βHSD2 did not reduce systemic prostacyclin production or accelerate atherosclerosis in mice, thereby avoiding the major cardiovascular side effects seen with systemic COX-2 inhibitors. Therefore, 11βHSD2 inhibition represents what we believe to be a novel approach for CRC chemoprevention and therapy by increasing tumor glucocorticoid activity, which in turn selectively blocks local COX-2 activity.

Colorectal cancer (CRC) is a leading cause of cancer death. COX-2–derived PGE2 promotes CRC progression (1), and inhibition of COX-2–derived PGE2 production by traditional NSAIDs or selective COX-2 inhibitors reduces the number and size of adenomas in familial adenomatous polyposis (FAP) patients and in mice with an autosomal-dominant heterozygous nonsense mutation of the mouse gene encoding adenomatous polyposis coli (Apc+/min mice; refs. 2–7). However, increased gastrointestinal side effects of traditional NSAIDs and increased cardiovascular risks of selective COX-2 inhibitors limit their utility in chemoprevention of CRC (8, 9).

Glucocorticoids mediate their antiinflammatory effects in part by inhibiting PG production. Unlike NSAIDs, which suppress PGE2 production by inhibition of COX enzymatic activity, glucocorticoids inhibit multiple steps in the PG cascade: inhibiting cytosolic phospholipase A2 (cPLA2) activity, which releases the COX substrate arachidonic acid, and inhibiting expression of both COX-2 and microsomal PGE synthase (mPGES-1), the terminal enzyme of COX-2–mediated PGE2 biosynthesis (10, 11). In addition to treatment of hematologic malignancies, glucocorticoids can inhibit solid tumor growth, regress tumor mass, and prevent metastasis by blocking angiogenesis (12–14). However, the undesirable side effects of immune suppression limit their application in cancer chemoprevention and chemotherapy.

In cultured cells, COX-2 expression and PGE2 production can be suppressed by glucocorticoids at concentrations as low as 10–9 M (15). Because levels of circulating glucocorticoids are approximately 10–6 to 10–7 M (cortisol in humans and corticosterone [CS] in rodents), COX-2 expression might be expected to be tonically suppressed by circulating glucocorticoids; however, COX-2 is constitutively expressed in human colonic adenomas and in Apc+/min mouse intestinal adenomas (5, 16). Normally, 11β–hydroxysteroid dehydrogenase type II (11βHSD2) inactivates intracellular glucocorticoids in the classic mineralocorticoid-responsive organs, kidney and intestine (particularly colon), in order to maintain the specificity of the mineralocorticoid receptor to activation by aldosterone (17). We have previously shown that pharmacologic inhibition of 11βHSD2 activity suppresses kidney cortex COX-2 expression by elevating intracellular levels of endogenous active glucocorticoids (18). In the present study, we determined that 11βHSD2 expression increased in parallel to COX-2 expression and activity in colonic adenomas. Furthermore, either genetic or pharmacologic inhibition of 11βHSD2 led to decreased COX-2 expression and activity in colonic adenomas and tumors and significantly suppressed adenoma and tumor growth. These findings suggest an important role for 11βHSD2 in regulation of COX-2 expression in colonic tumors and identify 11βHSD2 as a possible target for prevention and/or therapy of colorectal cancer.

Pharmacologic inhibition of 11βHSD2 suppressed adenoma COX-2 expression and growth in Apc+/min mice.
In Apc+/min mouse samples, increased expression of COX-2 and 11βHSD2 was evident in both adenoma stroma and epithelia and at the junction of tumor and normal tissue (Figure 3A). Administration of GA to Apc+/min mice for 8 wk significantly reduced total adenoma number and size as well as adenoma COX-2 and mPGES-1 expression (Figure 3, B–D).

Similar to 11βHSD2 knockdowns, pharmacologic inhibition of11βHSD2 activity with GA significantly enhanced low-dose CS–mediated inhibition of CT26 cell COX-2 expression (Figure 5A) and cell migration, to the same extent as a selective COX-2 inhibitor (Supplemental Figure 3B). GA also substantially reduced tumor COX-2 and PGE2 levels as well as tumor growth (Figure 5B) and number (Table 1). Inhibition of tumor growth was dose dependent, with maximal inhibition achieved at a GA concentration of 3 mg/kg/d (Supplemental Figure 4A), which is comparable to doses that have been safely administered to humans in long-term studies (19, 20). GA treatment also led to significant decreases in phosphorylated cPLA2 and mPGES-1 expression in tumors (Figure 5C). However, GA treatment had no effect on the expression of COX-1 or cytosolic PGE synthase (cPGES), a terminal enzyme in COX-1–mediated PGE2 biosynthesis (Figure 5C). The glucocorticoid receptor antagonist RU486 completely reversed GA inhibition of tumor COX-2 expression, PGE2 biosynthesis, and growth (Figure 5B), which indicates that glucocorticoid receptor activation mediated GA-induced inhibition of both COX-2 activity and CT26 tumor growth. GA treatment led to significant increases in CS levels with concomitant decreases in levels of the inactive CS metabolite 11-keto-corticosterone in kidney, colon, and tumors (Figure 5D). Most GA was found to be converted to its more active metabolite, glycyrrhetinic acid (GE), at 24 h after the last administration of GA (Supplemental Figure 5 and ref. 21). Furthermore, in CT26-derived tumors, GA had no additive effect on growth inhibition by the selective COX-2 inhibitor SC58236 (2 mg/kg/d i.p.; Supplemental Figure 4B). Therefore, COX-2 inhibition was an essential step in GA-mediated tumor suppression.

Inhibition of 11βHSD2 did not alter systemic PG production or promote atherogenesis. Although clinical trials have indicated that selective COX-2 inhibitors reduce colon adenoma formation in patients at high risk for CRC, these trials also highlighted the increased cardiovascular risk engendered by these agents (8, 26). Selective COX-2 inhibitors suppress endothelial COX-2–derived prostacyclin (PGI2) production without inhibition of COX-1–mediated prothrombotic platelet thromboxane A2 production, which increases the propensity for thrombogenesis and atherogenesis (27). Administration of COX-2 inhibitors to mice decreases urinary levels of the major PGI2 metabolite, 2,3-dinor-6-keto PGF1α (28). In contrast, administration of GA for 1 mo at concentrations up to 30 mg/kg/d did not inhibit urinary 2,3-dinor-6-keto PGF1α excretion or alter urinary 2,3-dinor thromboxane B2 excretion (Supplemental Figure 8A). Furthermore, in female apoE-null mice, a model of spontaneous atherosclerosis that recapitulates many features of human atherosclerosis (29), GA treatment (10 mg/kg/d i.p.) for 10 wk did not accelerate progression of atherosclerosis (Supplemental Figure 8B). GA treatment did not alter systolic blood pressure, and serum potassium was unchanged in mice administered concentrations of GA up to 30 mg/kg/d (data not shown). Therefore, in this model, chronic use of GA at tumor-suppressive concentrations did not induce the cardiovascular side effects previously reported with selective COX-2 inhibitors (8, 26).

Our present results indicate that tissue 11βHSD2 activity contributes to increased COX-2 expression in colorectal tumors and that inhibition of 11βHSD2 activity in vivo suppresses COX-2–derived PGE2 production and colorectal tumor growth as a result of increased intracellular active glucocorticoids (Figure 7). Recent studies have demonstrated a molecular link between COX-2–derived PGE2 and CRC progression (1). Clinical evidence indicates a 40%–50% reduction in CRC in individuals taking NSAIDs regularly, either in the context of sporadic CRC or in FAP patients (3, 30–32). However, long-term use of traditional NSAIDs increases gastrointestinal side effects. Reports estimate 10,000–20,000 deaths and 100,000 hospitalizations per year in the United States related to gastrointestinal complications induced by traditional NSAIDs (33). The antiinflammatory properties of NSAIDs are attributed to COX-2 inhibition, while their gastrointestinal side effects are attributed predominantly to COX-1 inhibition (6, 7, 34).

Proposed mechanism underlying 11βHSD2 activity and colorectal tumorigenesis. In tumor cells, glucocorticoids are converted to inactive keto-forms by 11βHSD2, reducing glucocorticoid receptor activation, while inhibition of 11βHSD2 activity (by GA and its analogs or by gene knockdown) leads to increased levels of intracellular active glucocorticoids. The consequent inhibition of cPLA2 activity and COX-2 and mPGES-1 expression results in blockade of PGE2 production and inhibition of tumor growth.

Selective COX-2 inhibitors have shown efficacy similar to that of traditional NSAIDs in treating acute and chronic inflammatory conditions and in reducing the number and size of adenomas in FAP patients and Apc+/min mice, with fewer gastrointestinal side effects (2, 4, 5). Therefore, COX-2 inhibitors were touted as promising agents for chemoprevention and chemotherapy of CRC. It has been suggested that the increased incidence of cardiovascular events associated with long-term use of selective COX-2 inhibitors is caused by the selective inhibition of COX-2–derived PGI2 production in endothelial cells (8, 9). Since platelets express only COX-1, COX-2 inhibition will not inhibit the production of COX-1–mediated prothrombotic platelet thromboxane A2 (27). In the current study, long-term treatment with a selective 11βHSD2 inhibitor did not promote atherogenesis in a mouse proatherogenic model, unlike what has been previously described with COX-2 inhibitors (35).

The physiological function of 11βHSD2 is to inactivate glucocorticoids, thereby preventing glucocorticoid-induced activation of mineralocorticoid receptors in aldosterone-responsive tissues such as the kidney and colon (15, 36). Inhibition of 11βHSD2 significantly inhibited CS metabolism and thereby increased levels of active glucocorticoid in colonic tumor (Figure 5D). Glucocorticoids are known to inhibit cell proliferation and induce cell differentiation through activation of glucocorticoid receptors. Although glucocorticoids have previously been used to treat certain hematological malignancies (37) and have been reported to inhibit the growth of colon cancer cell lines and to inhibit CRC hepatic metastasis (12, 13), systemic administration of glucocorticoids is not suitable for chemoprevention and chemotherapy of CRC because of immunosuppression and other systemic side effects.

Recently, the potential role of 11βHSD2 in tumorigenesis has attracted interest. Expression of 11βHSD2 increases in some tumors and tumor cell lines (38–40). In MCF-7 breast cancer cells, glucocorticoid-induced inhibition of cell proliferation is attenuated by 11βHSD2 (41). The results of the present study indicate that a major mechanism for an antitumor effect of 11βHSD2 inhibition in colonic adenomas and carcinomas is the inhibition of COX-2 expression and production of COX-2–dependent PGs by increasing local concentrations of glucocorticoids, and that this COX-2 inhibition will markedly inhibit tumorigenesis.

We propose that inhibition of 11βHSD2 activity may provide a new target for chemoprevention and/or adjunctive therapy for CRC, particularly for patients with increased risk, such as FAP patients, because of the following advantages. First, selectively decreased COX-2, but not COX-1, mediated PG production. Therefore, inhibition of 11βHSD2 activity has the beneficial effects of traditional NSAIDs to prevent and regress CRC without the gastrointestinal side effects associated with COX-1 inhibition. Second, physiologic 11βHSD2 expression is largely restricted to colon and kidney. Therefore, inhibition of 11βHSD2 activity is not expected to incur the cardiovascular risk posed by COX-2 inhibitors that suppress COX-2–derived PGI2 production in vascular endothelial cells. Finally, increased levels of intracellular active glucocorticoids were observed only in tissues with elevated levels of 11βHSD2 expression. Inhibition of 11βHSD2 will not produce immunosuppression or other systemic side effects of conventional glucocorticoid therapy. It is also possible that increased intracellular active endogenous glucocorticoids may also inhibit colorectal tumorigenesis through non–PG-mediated pathways, such as stimulation of p53-dependent pathways (42).

Tissue PG levels are determined by both biosynthesis and catabolism. A key enzyme in PG catabolism, 15-hydroxyprostaglandin dehydrogenase, has been reported to decrease in human colorectal cancer and in Apc+/min mouse intestinal adenomas (43). Studies have indicated that glucocorticoids can induce 15-hydroxyprostaglandin in A549 human lung adenocarcinoma cells (44). Therefore, it is possible that increased intracellular active endogenous glucocorticoids may inhibit colorectal tumorigenesis not only by inhibiting PG synthesis, but also by enhancing PG degradation.

GA and its analogs are excellent prototypes for 11βHSD2 inhibitors. GA is a natural compound contained in licorice, a natural botanical antiinflammatory agent and a powerful 11βHSD2 inhibitor. Long-term excessive ingestion of licorice has been reported to induce hypokalemia and elevation of blood pressure in a subset of people (20). Although these side effects were not seen with GA treatment in our experimental animals, nor have they been reported as a substantial limitation in studies of humans treated daily with comparable doses of GA for extended periods (19, 45), we did observe that the concentrations of GA used in our studies increased levels of active CS in the kidney (Figure 5D). Therefore, it is likely that if comparable doses of GA were used in humans, some percentage of patients would develop hypertension and/or hypokalemia, requiring treatment with the potassium-sparing diuretic amiloride. However, the need to monitor for these potential side effects should not necessarily preclude the use of 11βHSD2 inhibition as a strategy to inhibit colorectal tumor COX-2 expression and growth.

Apc+/min mouse model.
The germline mutations in the APC gene lead to FAP, and inactivation of APC is also found in most sporadic colorectal cancers (5). Apc+/min mice have an autosomal-dominant heterozygous nonsense mutation of the mouse Apc gene, homologous to human germline and somatic APC mutations, and develop adenomas to a grossly detectable size within a few months. Male Apc+/min mice (5 wk old) were obtained from The Jackson Laboratory and treated with either vehicle (water) or GA (30 mg/kg/d i.p.) for 8 wk. Under anesthesia with Nembutal (60 mg/kg i.p.; Abbot Laboratories), the entire intestine was dissected, flushed thoroughly with ice-cold PBS (pH 7.4), and then filled with fixative (15). The intestine was transferred to 70% ethanol for 24 h, opened longitudinally, and examined using a dissecting microscope to count polyps in a blinded fashion. The tumor diameter was measured with a digital caliper. After tumors were counted, intestinal tissues were processed for paraffin embedding. All animal experiments were performed according to animal care guidelines and were approved by the Vanderbilt University IACUC.

Metastatic tumor growth. Male BALB/c mice (8 wk old; 18–20 g) received a single tail vein injection of wild-type CT26 cells or 11βHSD2 knockdown CT26 cells (2.5 × 104 cells in 0.5 ml medium). Mice receiving CT26 cells were administered vehicle (water) or GA (10 mg/kg/d i.p.) initiated 1 d prior to cell injection. We sacrificed 6 mice from each group 4 wk after injection and kept the remaining mice until death to generate survival curves. The lungs were removed, weighed, and fixed in fixative or kept in PBS, after which the metastatic lung tumors were counted and tumor volumes measured as described above (47).

GA and blood pressure and atherosclerosis in Apoe–/– mice.
Female Apoe–/– mice on the C57BL/6 background were obtained from The Jackson Laboratory. The mice were fed a high-fat diet (0.2% cholesterol, 21% saturated fat, formula TD88173; Harlan Teklad) from 6 wk of age and treated with vehicle (water) or GA (10 mg/kg/d, i.p.) for 10 wk. Blood pressure was measured by tail-cuff method 1 wk before sacrifice. Under anesthesia with Nembutal (60 mg/kg i.p.; Abbot Laboratories), the hearts and proximal aortae were dissected and placed in formalin, paraffin embedded, and cut (10 μm) serially from the proximal aorta beginning at the end of the aortic sinus. The sections were stained with Oil-Red-O and counterstained with hematoxylin (Sigma-Aldrich), and quantitative analysis of lesions was performed on 15 sections from each animal (48). The remaining aortae were dissected out. En face preparations were longitudinally opened, pinned flat, stained with Sudan IV, photographed, and analyzed using the BIOQUANT true-color windows system (R&M Biometrics). The lesion was expressed as percentage of en face aorta surface. The analysis was performed by the same investigator blinded to the study groups.

Establishment of shRNA stable cell lines. We inserted 2 unique 19-bp oligos derived from 11βHSD2 mRNA, 5′-GGAGACAGGTAAGAAACTG-3′ and 5′-GGTGAACTTCTTTGGTGCA-3′, which targeted 11βHSD2 exon 2 and exon 3, as well as negative control, 5′-GCGCGCTTTGTAGGATTCG-3′ (Oligoengine), into pSuper.neo-GFP vector (Oligoengine), which provided the shRNA backbone and were named 11βHSD2/shRNA2, 11βHSD2/shRNA3, and shscramble, respectively. After sequence confirmation of each construct, we transfected 11βHSD2/shRNA2, 11βHSD2/shRNA3, and shscramble into subconfluent CT26 cells using Effectene (Qiagen) to produce stable cell lines designated CT26shRNA2, CT26shRNA3, and CT26shscramble. After 24 h, DMEM containing 10% fetal bovine serum and 1 mg/ml G418 was used to select for G418-resistant clones. After 1 wk of G418-selective culture, the remaining cells were resuspended and reselected by fluorescence-activated cell sorting for the presence of GFP in the vector. Single clones were chosen by growing the reselected transfected cells under 1 mg/ml G418 using serial dilution.

Transient transfection of 11βHSD2. Expression vector pCMV-SPORT6, containing the entire open reading frame of mouse 11βHSD2 cDNA, was obtained from Open Biosystems. A total of 0.8 μg 11βHSD2 construct or empty pCMV-SPORTS vector was used for transient transfection into subconfluent CT26 cells using Effectene (Qiagen). The cells were used 48–72 h after transfection.

RNA isolation and quantitative real-time PCR. Total RNA was isolated from CT26 cells, stable cell lines, and human colonic adenomas and colon tissues using TRIzol reagents (Invitrogen) according to the manufacturer’s instructions. Quantitative PCR was performed using the iCycler iQ Real Time PCR detection System (Bio-Rad). The primers used for mouse 11βHSD2 were 5′-GCCACTCTTGCGTCACTC-3′ (forward) and 5′-AGCCGAATGTGTCCATAAGC-3′ (reverse). The primers used for mouse GAPDH were 5′-CCAGAACATCATCCCTGCAT-3′ (forward) and 5′-GTTCAGCTCTGGGATGACCTT-3′ (reverse). The primers used for human 111βHSD2 (Hs00388669) and β-actin (Hs99999903) were from Applied Biosystems.

Cell migration and cell proliferation assays. CT26, HCA-7, and HT-29 cells were grown in DMEM supplemented with 4,500 mg/l glucose, 2 mM l-glutamine, 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin in 5% CO2 and 95% air at 37°C. Cell migration assays were performed in Transwells (8 μm; Corning Costar Co.) as previously described (49). The underside of each Transwell was precoated with collagen I overnight at 4°C, and the filter was subsequently blocked with 1% bovine serum albumin for 1 h at 37°C to inhibit nonspecific migration. CT26 cells were cultured in serum-free medium overnight and then treated with 10 μM GA, 10 nM CS, GA plus CS, or 10 μM of the COX-2 inhibitor SC58236 for 3 h. GA was added 30 min before addition of CS. The cell suspensions (100 μl, 1 × 106 cells/ml) were added to the wells, and cells were allowed to migrate into the matrix coated on the underside of the Transwell for 2.5 h. Cells on the top of the filter were removed, the filter was fixed in 4% formaldehyde and stained with 1% crystal violet, and migrating cells were counted using an image analysis system (R & M Biometrics). For cell proliferation assays, HCA-7 or HT-29 cells were cultured in 24-well plates (1 ml, 1 × 105 cells/well) in medium with or without the COX-2 inhibitors SC58236 (25 μM) or NS-398 (5 and 25 μM). The medium was changed every other day, and cells were counted 2, 4, and 6 d after seeding by an investigator blinded to the study groups.

Preservation and fixation of mouse and human tissues. Tissues from human colonic adenoma resections were immersed in formalin and processed for paraffin embedding. The mice bearing tumors were anesthetized with Nembutal (60 mg/kg i.p.; Abbot Laboratories), given heparin (1,000 U/kg i.p.) to minimize coagulation, and perfused with fixative through the aortic trunk (15). After fixation, the tumor tissues were dehydrated, paraffin embedded, sectioned, and mounted on glass slides. The samples used in this study were from the Tennessee Colorectal Polyp Study (TCPS) protocol that was approved by the Institutional Review Board of Vanderbilt Medical Center, and written, informed consent was obtained from all participants.

Immunofluorescence/immunohistochemistry staining and quantitative image analysis. For immunofluorescence, frozen sections (without fixation) were first washed with PBS, then incubated with rat anti-murine CD31 antibody (BD Biosciences — Pharmingen) for 1 h and washed again with PBS. Washed sections were treated with fluorescence-conjugated secondary antibodies for 1 h. For paraffin-embedded tissue sections, the slides were deparaffinized, rehydrated, and stained with different antibodies, as previously described (15). Based on the distinctive density and color of immunostaining in video images, the number, size, and position of stained cells were quantified using the BIOQUANT true-color windows system (R & M Biometrics) as previously described (50).

Western blot analysis. Total CT26 cell lysate and total CT26 tumor lysate were used for Western blot analysis as described previously (51).

Measurement of prostanoids. Tumor PGE2 levels were quantified by gas chromatographic/negative ion chemical ionization mass spectrometric assays using stable isotope dilution (52). PGE2 concentration in the culture medium was measured using Prostaglandin E2 Express EIA Kid according to the manufacturer’s instruction (Cayman Chemical). The cells were first cultured in the medium with 10 nM CS. After 24 h, the medium was changed and incubated for an additional 1 h. The medium was collected for determination of PGE2 concentration, and the cells were lysed for protein assay.

Measurement of CS, 11-keto-corticosterone, GA, and GE. At 24 h after the last i.p. injection of GA, the animals were sacrificed, and kidneys, colons, and tumors were collected and stored at –80°C. Tissue levels of CS, 11-keto-corticosterone, GA, and GE were measured using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (53). GA and GE were from Sigma-Aldrich, CS was from ICN Biomedicals, and 11-keto-corticosterone was from Steroids.

To investigate the inhibition of CT26 cell 11βHSD2 activity by GA, cells were washed with serum-free medium, then treated with 100 nM CS alone or 100 nM CS plus 10 μM GA for 1 h, and medium was collected for determination of CS and 11-keto-corticosterone levels. GA was added 30 min before addition of CS. In the absence of any inhibitor, parental CT26 converted approximately 40% of the administered CS to the metabolite, 11-keto CS, indicating active 11βHSD2 activity in these cells. The inhibition of 11βHSD2 activity in CT26 by 11βHSD2 knockdown was also investigated using CT26shRNA3–31 and CT26shRNA2–9 cells. We expressed 11βHSD2 activity as timed production of 11-keto-corticosterone per mg protein.

Statistics. All values are presented as mean ± SD. Fisher exact test, ANOVA, and 2-tailed Student’s t test with Bonferroni correction were used for statistical analysis. A P value less than 0.05 was considered significant.